Employing relaxed stringency hybridization conditions with v-erbB as probe, we previously had identified a novel member of the erbB/EGF receptor gene family in the human genome amplified in a mammary carcinoma. In a series of human mammary tumor cell lines, transcript analysis demonstrated elevated expression levels of erbB-2 ranging from 8- to 128-fold above those of normal controls in 8 out of 16 cases. An aberrantly sized erbB-2 transcript was not detected in these cell lines. Immunoblot analysis indicated elevated levels of the 185-kd product of erbB-2 expressed by these cells. In four lines, erbB-2 gene amplification in the absence of an apparent gene rearrangement was demonstrated. Amplified gene copies in a representative cell line, SK-BR-3, were localized in an aberrant chromosomal location by in situ hybridization. In four additional cell lines, 4-to 8-fold erbB-2 mRNA overexpression was observed in the absence of gene amplification. In a representative cell line, 7R-75-1, normal chromosomal location of erbB-2 was determined. Moreover, gene amplification of erbB-2 was observed in 10% of human mammary tumor tissues analyzed. These findings linked overexpression of an apparently normal erbB-2 gene product with human mammary neoplasia. In order to assess the transforming potential of this growth factor receptor-like gene, we introduced the normal coding sequence of erbB-2 in NIH/3T3 cells by DNA transfection expressing the gene product at different expression levels. Under SV40 promoter, the gene lacked transforming activity despite expression of erbB-2 protein levels. A five to tenfold increase in its expression under LTR influence was associated with activation of erbB-2 as a potent oncogene. The higher levels of erbB-2 protein were observed in mammary tumor cell lines with erbB-2 gene amplification.